Spontaneous allelic variant in deafness-blindness gene Ush1g resulting in an expanded phenotype.

Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1gbw/bw null mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell's kinocilial links. Consistent with other Usher type 1 models, Ush1gbw/bw mice had no substantial retinal degeneration compared with Ush1gbw /+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene.

GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region.

GIPC3 has been implicated in auditory function. Here, we establish that GIPC3 is initially localized to the cytoplasm of inner and outer hair cells of the cochlea and then is increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at 1 month of age. Cuticular plates of Gipc3KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks and the cuticular plate. Several of immunoprecipitated proteins contained GIPC family consensus PDZ-binding motifs (PBMs), including MYO18A, which bound directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell junction proteins to shape the cuticular plate.

Control of stereocilia length during development of hair bundles.

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later-in early stage IV-and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J, and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction's role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.

GIPC3 couples to MYO6 and PDZ domain proteins and shapes the hair cell apical region.

GIPC3 has been implicated in auditory function. Initially localized to the cytoplasm of inner and outer hair cells of the cochlea, GIPC3 increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3 KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at one month of age. Cuticular plates of Gipc3 KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3 KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks, and the cuticular plate. Several of immunoprecipitated proteins contained GIPC-family consensus PDZ binding motifs (PBMs), including MYO18A, which binds directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell-junction proteins to shape the cuticular plate. Summary statement: The PDZ-domain protein GIPC3 couples the molecular motors MYO6 and MYO18A to actin cytoskeleton structures in hair cells. GIPC3 is necessary for shaping the hair cell’s cuticular plate and hence the arrangement of the stereocilia in the hair bundle.

ANKRD24 organizes TRIOBP to reinforce stereocilia insertion points.

The stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell's cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed-TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience.

A cryo-tomography-based volumetric model of the actin core of mouse vestibular hair cell stereocilia lacking plastin 1.

Electron cryo-tomography allows for high-resolution imaging of stereocilia in their native state. Because their actin filaments have a higher degree of order, we imaged stereocilia from mice lacking the actin crosslinker plastin 1 (PLS1). We found that while stereocilia actin filaments run 13 nm apart in parallel for long distances, there were gaps of significant size that were stochastically distributed throughout the actin core. Actin crosslinkers were distributed through the stereocilium, but did not occupy all possible binding sites. At stereocilia tips, protein density extended beyond actin filaments, especially on the side of the tip where a tip link is expected to anchor. Along the shaft, repeating density was observed that corresponds to actin-to-membrane connectors. In the taper region, most actin filaments terminated near the plasma membrane. The remaining filaments twisted together to make a tighter bundle than was present in the shaft region; the spacing between them decreased from 13 nm to 9 nm, and the apparent filament diameter decreased from 6.4 to 4.8 nm. Our models illustrate detailed features of distinct structural domains that are present within the stereocilium.

Mechanotransduction-Dependent Control of Stereocilia Dimensions and Row Identity in Inner Hair Cells.

Actin-rich structures, like stereocilia and microvilli, are assembled with precise control of length, diameter, and relative spacing. By quantifying actin-core dimensions of stereocilia from phalloidin-labeled mouse cochleas, we demonstrated that inner hair cell stereocilia developed in specific stages, where a widening phase is sandwiched between two lengthening phases. Moreover, widening of the second-tallest stereocilia rank (row 2) occurred simultaneously with the appearance of mechanotransduction. Correspondingly, Tmc1KO/KO;Tmc2KO/KO or TmieKO/KO hair cells, which lack transduction, have significantly altered stereocilia lengths and diameters, including a narrowed row 2. EPS8 and the short splice isoform of MYO15A, identity markers for mature row 1 (the tallest row), lost their row exclusivity in transduction mutants. GNAI3, another member of the mature row 1 complex, accumulated at mutant row 1 tips at considerably lower levels than in wild-type bundles. Alterations in stereocilia dimensions and in EPS8 distribution seen in transduction mutants were mimicked by block of transduction channels of cochlear explants in culture. In addition, proteins normally concentrated at mature row 2 tips were also distributed differently in transduction mutants; the heterodimeric capping protein subunit CAPZB and its partner TWF2 never concentrated at row 2 tips like they do in wild-type bundles. The altered distribution of marker proteins in transduction mutants was accompanied by increased variability in stereocilia length. Transduction channels thus specify and maintain row identity, control addition of new actin filaments to increase stereocilia diameter, and coordinate stereocilia height within rows.

Single-cell proteomics reveals changes in expression during hair-cell development.

Hearing and balance rely on small sensory hair cells that reside in the inner ear. To explore dynamic changes in the abundant proteins present in differentiating hair cells, we used nanoliter-scale shotgun mass spectrometry of single cells, each ~1 picoliter, from utricles of embryonic day 15 chickens. We identified unique constellations of proteins or protein groups from presumptive hair cells and from progenitor cells. The single-cell proteomes enabled the de novo reconstruction of a developmental trajectory using protein expression levels, revealing proteins that greatly increased in expression during differentiation of hair cells (e.g., OCM, CRABP1, GPX2, AK1, GSTO1) and those that decreased during differentiation (e.g., TMSB4X, AGR3). Complementary single-cell transcriptome profiling showed corresponding changes in mRNA during maturation of hair cells. Single-cell proteomics data thus can be mined to reveal features of cellular development that may be missed with transcriptomics.

Electron cryo-tomography of vestibular hair-cell stereocilia.

High-resolution imaging of hair-cell stereocilia of the inner ear has contributed substantially to our understanding of auditory and vestibular function. To provide three-dimensional views of the structure of stereocilia cytoskeleton and membranes, we developed a method for rapidly freezing unfixed stereocilia on electron microscopy grids, which allowed subsequent 3D imaging by electron cryo-tomography. Structures of stereocilia tips, shafts, and tapers were revealed, demonstrating that the actin paracrystal was not perfectly ordered. This sample-preparation and imaging procedure will allow for examination of structural features of stereocilia in a near-native state.

Molecular Composition of Vestibular Hair Bundles.

The vertebrate hair bundle, responsible for transduction of mechanical signals into receptor potentials in sensory hair cells, is an evolutionary masterpiece. Composed of actin-filled stereocilia of precisely regulated length, width, and number, the structure of the hair bundle is optimized for sensing auditory and vestibular stimuli. Recent developments in identifying the lipids and proteins constituting the hair bundle, obtained through genetics, biochemistry, and imaging, now permit a description of the consensus composition of vestibular bundles of mouse, rat, and chick.

Mass spectrometry quantitation of proteins from small pools of developing auditory and vestibular cells.

Hair cells of the inner ear undergo postnatal development that leads to formation of their sensory organelles, synaptic machinery, and in the case of cochlear outer hair cells, their electromotile mechanism. To examine how the proteome changes over development from postnatal days 0 through 7, we isolated pools of 5000 Pou4f3-Gfp positive or negative cells from the cochlea or utricles; these cell pools were analysed by data-dependent and data-independent acquisition (DDA and DIA) mass spectrometry. DDA data were used to generate spectral libraries, which enabled identification and accurate quantitation of specific proteins using the DIA datasets. DIA measurements were extremely sensitive; we were able to detect proteins present at less than one part in 100,000 from only 312 hair cells. The DDA and DIA datasets will be valuable for accurately quantifying proteins in hair cells and non-hair cells over this developmental window.

ELMOD1 Stimulates ARF6-GTP Hydrolysis to Stabilize Apical Structures in Developing Vestibular Hair Cells.

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ADP-ribosylation factor (ARF) small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMO domain-containing protein 1 (ELMOD1), a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we analyzed mice of both sexes from a strain lacking functional ELMOD1 [roundabout (rda)]. In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5; large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cell's apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles compared with controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle.SIGNIFICANCE STATEMENT Assembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia.

Heterodimeric capping protein is required for stereocilia length and width regulation.

Control of the dimensions of actin-rich processes like filopodia, lamellipodia, microvilli, and stereocilia requires the coordinated activity of many proteins. Each of these actin structures relies on heterodimeric capping protein (CAPZ), which blocks actin polymerization at barbed ends. Because dimension control of the inner ear's stereocilia is particularly precise, we studied the CAPZB subunit in hair cells. CAPZB, present at ∼100 copies per stereocilium, concentrated at stereocilia tips as hair cell development progressed, similar to the CAPZB-interacting protein TWF2. We deleted Capzb specifically in hair cells using Atoh1-Cre, which eliminated auditory and vestibular function. Capzb-null stereocilia initially developed normally but later shortened and disappeared; surprisingly, stereocilia width decreased concomitantly with length. CAPZB2 expressed by in utero electroporation prevented normal elongation of vestibular stereocilia and irregularly widened them. Together, these results suggest that capping protein participates in stereocilia widening by preventing newly elongating actin filaments from depolymerizing.

Corrigendum: Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like.

This corrects the article DOI: 10.1038/ncomms10833.

Integration of Tmc1/2 into the mechanotransduction complex in zebrafish hair cells is regulated by Transmembrane O-methyltransferase (Tomt).

Transmembrane O-methyltransferase (TOMT/LRTOMT) is responsible for non-syndromic deafness DFNB63. However, the specific defects that lead to hearing loss have not been described. Using a zebrafish model of DFNB63, we show that the auditory and vestibular phenotypes are due to a lack of mechanotransduction (MET) in Tomt-deficient hair cells. GFP-tagged Tomt is enriched in the Golgi of hair cells, suggesting that Tomt might regulate the trafficking of other MET components to the hair bundle. We found that Tmc1/2 proteins are specifically excluded from the hair bundle in tomt mutants, whereas other MET complex proteins can still localize to the bundle. Furthermore, mouse TOMT and TMC1 can directly interact in HEK 293 cells, and this interaction is modulated by His183 in TOMT. Thus, we propose a model of MET complex assembly where Tomt and the Tmcs interact within the secretory pathway to traffic Tmc proteins to the hair bundle.

Plastin 1 widens stereocilia by transforming actin filament packing from hexagonal to liquid.

With their essential role in inner ear function, stereocilia of sensory hair cells demonstrate the importance of cellular actin protrusions. Actin packing in stereocilia is mediated by cross-linkers of the plastin, fascin, and espin families. Although mice lacking espin (ESPN) have no vestibular or auditory function, we found that mice that either lacked plastin 1 (PLS1) or had nonfunctional fascin 2 (FSCN2) had reduced inner ear function, with double-mutant mice most strongly affected. Targeted mass spectrometry indicated that PLS1 was the most abundant cross-linker in vestibular stereocilia and the second most abundant protein overall; ESPN only accounted for ∼15% of the total cross-linkers in bundles. Mouse utricle stereocilia lacking PLS1 were shorter and thinner than wild-type stereocilia. Surprisingly, although wild-type stereocilia had random liquid packing of their actin filaments, stereocilia lacking PLS1 had orderly hexagonal packing. Although all three cross-linkers are required for stereocilia structure and function, PLS1 biases actin toward liquid packing, which allows stereocilia to grow to a greater diameter.

Neuroplastin Isoform Np55 Is Expressed in the Stereocilia of Outer Hair Cells and Required for Normal Outer Hair Cell Function.

UNLABELLED: Neuroplastin (Nptn) is a member of the Ig superfamily and is expressed in two isoforms, Np55 and Np65. Np65 regulates synaptic transmission but the function of Np55 is unknown. In an N-ethyl-N-nitrosaurea mutagenesis screen, we have now generated a mouse line with an Nptn mutation that causes deafness. We show that Np55 is expressed in stereocilia of outer hair cells (OHCs) but not inner hair cells and affects interactions of stereocilia with the tectorial membrane. In vivo vibrometry demonstrates that cochlear amplification is absent in Nptn mutant mice, which is consistent with the failure of OHC stereocilia to maintain stable interactions with the tectorial membrane. Hair bundles show morphological defects as the mutant mice age and while mechanotransduction currents can be evoked in early postnatal hair cells, cochlea microphonics recordings indicate that mechanontransduction is affected as the mutant mice age. We thus conclude that differential splicing leads to functional diversification of Nptn, where Np55 is essential for OHC function, while Np65 is implicated in the regulation of synaptic function. SIGNIFICANCE STATEMENT: Amplification of input sound signals, which is needed for the auditory sense organ to detect sounds over a wide intensity range, depends on mechanical coupling of outer hair cells to the tectorial membrane. The current study shows that neuroplastin, a member of the Ig superfamily, which has previously been linked to the regulation of synaptic plasticity, is critical to maintain a stable mechanical link of outer hair cells with the tectorial membrane. In vivo recordings demonstrate that neuroplastin is essential for sound amplification and that mutation in neuroplastin leads to auditory impairment in mice.

PDZD7-MYO7A complex identified in enriched stereocilia membranes.

While more than 70 genes have been linked to deafness, most of which are expressed in mechanosensory hair cells of the inner ear, a challenge has been to link these genes into molecular pathways. One example is Myo7a (myosin VIIA), in which deafness mutations affect the development and function of the mechanically sensitive stereocilia of hair cells. We describe here a procedure for the isolation of low-abundance protein complexes from stereocilia membrane fractions. Using this procedure, combined with identification and quantitation of proteins with mass spectrometry, we demonstrate that MYO7A forms a complex with PDZD7, a paralog of USH1C and DFNB31. MYO7A and PDZD7 interact in tissue-culture cells, and co-localize to the ankle-link region of stereocilia in wild-type but not Myo7a mutant mice. Our data thus describe a new paradigm for the interrogation of low-abundance protein complexes in hair cell stereocilia and establish an unanticipated link between MYO7A and PDZD7.

Annexin A5 is the Most Abundant Membrane-Associated Protein in Stereocilia but is Dispensable for Hair-Bundle Development and Function.

The phospholipid- and Ca(2+)-binding protein annexin A5 (ANXA5) is the most abundant membrane-associated protein of ~P23 mouse vestibular hair bundles, the inner ear's sensory organelle. Using quantitative mass spectrometry, we estimated that ANXA5 accounts for ~15,000 copies per stereocilium, or ~2% of the total protein there. Although seven other annexin genes are expressed in mouse utricles, mass spectrometry showed that none were present at levels near ANXA5 in bundles and none were upregulated in stereocilia of Anxa5(-/-) mice. Annexins have been proposed to mediate Ca(2+)-dependent repair of membrane lesions, which could be part of the repair mechanism in hair cells after noise damage. Nevertheless, mature Anxa5(-/-) mice not only have normal hearing and balance function, but following noise exposure, they are identical to wild-type mice in their temporary or permanent changes in hearing sensitivity. We suggest that despite the unusually high levels of ANXA5 in bundles, it does not play a role in the bundle's key function, mechanotransduction, at least until after two months of age in the cochlea and six months of age in the vestibular system. These results reinforce the lack of correlation between abundance of a protein in a specific compartment or cellular structure and its functional significance.

Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like.

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell.